Bilirubin metabolism, Hemolytic anemia-classification and lab diagnosis

1. Bilirubin metabolism, Hemolytic
anemia - classification and lab
diagnosis
Speaker:
Dr Harshita Aggarwal
Moderator:
Dr Kshitija Mittal
Dr Simranjeet Kaur

2. BILIRUBIN PRODUCTION
• Bilirubin is a tetrapyrrole pigment, a breakdown (catabolic) product of heme (ferroprotoporphyrin IX).
• After ~ 120 days in the circulation, RBCs are degraded by the reticuloendothelial (RE) cells, primarily
in the spleen, liver and bone marrow.
• An adult human normally destroys about 200 billion erythrocytes per day.
• Therefore, a 70 kg healthy human turns over approx. 6gm of hemoglobin daily.

3. • All products of hemoglobin breakdown are reused.
• Globin gets split from heme and returns to body’s metabolic “protein pool” where its
amino acids are reutilised.
• Iron(Fe3+) released from the heme is utilised by the cell (essential component of many
enzymes), exported (via ferroportin) or stored as ferritin.
• The iron-free porphyrin portion of heme undergoes subsequent biochemical reactions
before getting converted to yellow-pigment bilirubin.

4. • First reaction – catalysed by the microsomal
enzyme heme oxygenase, cleaves the alpha-
bridge of the iron-free porphyrin group and opens
the heme ring.
• The iron of heme that reaches heme oxygenase
gets oxidized to its ferric form (hemin/Fe3+)
which further enters the iron pool.
• The end products thus formed are biliverdin,
carbon monoxide and hemin.
• Biliverdin is a green-coloured pigment excreted
by birds and amphibians.
• Second reaction – In humans, this reaction is
catalysed by the cytosolic enzyme biliverdin
reductase, reduces central methylene bridge of
biliverdin to methyl group, producing yellow
pigment bilirubin.

5. • Since 1 gm of hemoglobin yields about 35mg of bilirubin, adult humans form 250-350
mg of bilirubin per day.
• About 70-80% of the bilirubin produced each day is derived from the breakdown of
hemoglobin in senescent red blood cells.
• The remainder comes from
- prematurely destroyed erythroid cells in bone marrow
- turnover of hemoproteins such as myoglobin and cytochromes

6. It is interesting to note that conversion of heme
to bilirubin by reticuloendothelial cells can be
observed visually as the purple color of the heme
in a hematoma slowly converts to the yellow
pigment of bilirubin

7. BILIRUBIN METABOLISM
• Once bilirubin is released by the RE cells, it appears in the plasma (Unconjugated form)
• Normal plasma bilirubin is 0.2-1.0 mg/dl
• Unconjugated bilirubin is insoluble in water due to tight internal hydrogen bonding.
To be transported in blood, bilirubin must be solubilized.
This solubilisation is accomplished by the reversible binding of bilirubin to albumin.
Binds approx. 25mg of
bilirubin/100 ml of
plasma

8. • Further metabolism of bilirubin occurs primarily in liver
• Hepatic catabolism of bilirubin takes place in three distinct but interrelated steps:
Hepatocellular
uptake and
intracellular
binding
Bilirubin
conjugation with
glucuronic acid
Biliary excretion

9. Hepatocellular uptake and intracellular binding:
• Bilirubin-albumin complex is transported to liver.
• Bilirubin is removed from albumin and taken up
at the sinusoidal surface of hepatocytes by carrier-
mediated membrane transport system.
• Within the hepatocyte, bilirubin binds to cytosolic
protein i.e. glutathione-S-transferases/ligandin, it
prevents bilirubin from re-entering the
bloodstream.
Conjugation with glucuronate :
• Bilirubin is conjugated with one or two
glucuronic acid moieties from UDP-glucoronate
• Catalyzed by bilirubin-specific endoplasmic
reticulum enzyme i.e. UDP-
glucuronosyltransferase which form bilirubin
mono- and diglucuronide, in stepwise manner.

10. Conjugation is obligatory for excretion of bilirubin across the bile canalicular membrane into
bile, as it converts bilirubin into polar (water-soluble) form
Normal conjugated bilirubin levels are ≤ 0.25mg/dl
Biliary excretion:
• Secretion of conjugated bilirubin across the bile canalicular membrane into the bile is an
active transport mechanism
• Rate-limiting step for the entire process of hepatic bilirubin metabolism
• Mediated by ATP dependant canalicular membrane protein called multispecific organic anion
transporter (MOAT).

11. Extrahepatic Aspect Of Bilirubin Disposition:
Bilirubin in the gut:
• Following secretion into the bile, conjugated bilirubin reaches the duodenum via the bile
duct and passes down the gut (terminal ileum and large intestine) without reabsorption by
the intestinal mucosa.
• The glucuronosyl moieties are removed by specific bacterial ẞ-glucuronidases
• Bilirubin further reduced by the bacterial flora to a group of colorless compounds, referred
to as urobilinogens.
• Approx 10 to 20 per cent of the urobilinogen is reabsorbed from terminal ileum and large
intestine into the portal vein and is re-excreted by the liver into the bile, thus returning to the
bowel.
• This is called the enterohepatic circulation of bile pigments or enterohepatic
urobilinogen cycle.

12. REF: Enterohepatic cycling of bilirubin as
a cause of ‘black’ pigment gallstones in
adult life – European journal of clinical
investigation

13. Renal excretion of bilirubin:
• Rest of the urobilinogen passes into the systemic circulation and excreted by the kidneys after
oxidizing it into coloured urobilins.
• Under abnormal condition like excessive bile pigment production or liver disease disrupting intra-
hepatic cycle, urobilinogen may also excreted in urine

14. Refrence: Lippincott
Illustrated reviews of
Biochemistry 7th edition

15. Ref: de Gruchy’s clinical
haematology in medial
practice

16. DISORDERS OF BILIRUBIN METABOLISM
CAUSES :
 Either production of more bilirubin than normal liver can excrete
 From failure of damaged liver to excrete normal amount of bilirubin
 Or obstruction of extra-hepatic biliary tree in the absence of any hepatic damage
• Resulting in hyperbilirubinemia → >1mg/dl of total bilirubin concentration in blood
• Conjugated bilirubin levels, also called as direct bilirubin levels > 0.25mg/dl is considered as conjugated
hyperbilirubinemia
• Difference between total bilirubin and direct bilirubin is known as indirect bilirubin or unconjugated
bilirubin → > 0.8mg/dl is considered as unconjugated hyperbilirubinemia
• When blood concentration of total bilirubin reaches to 2-2.5mg/dl → diffuses into tissues → yellow
discoloration, condition termed as Jaundice or Icterus.
• The condition can arise due to either unconjugated hyperbilirubinemia or conjugated hyperbilirubinemia or
both.

17. Classification of hyperbilirubinemia based on unconjugated and conjugated
causes of hyperbilirubinemia :
Unconjugated Conjugated
Hemolytic anemias Obstruction of biliary tree
Neonatal physiological jaundice Dubin-Johnson syndrome
Crigler-Najjar syndromes type 1 & 2 Rotor syndrome
Gilbert syndrome Viral hepatitis
Liver diseases

18. Refrence : Lippincott Illustrated Reviews Biochemistry 7th edition
UNCONJUGATED
HYERBILIRUBIN-
EMIA
CONJUGATED
HYERBILIRUBIN-
EMIA
CONJ/UNCONJUGATED
HYERBILIRUBINEMIA

19. Classification on basis of Pre-hepatic, Hepatic & Post-hepatic causes of hyperbilirubinemia
PRE-HEPATIC HEPATIC POST-HEPATIC
Excessive production
• Hemolytic anemias
Decreased hepatic
uptake/ conjugation/
excretion
• Viral hepatitis
• Alcoholic liver disease
• Neonatal physiological
jaundice
• Crigler-Najjar syndrome I, II
• Gilbert syndrome
• Dubin-Johnson, Rotor
syndrome
• Drugs and toxins
Extra hepatic biliary
obstruction
• Gall stones
• Ca head pancreas, Ca gall
bladder, cholangiocarcinoma
• Primary sclerosing
cholangitis
• Extrahepatic biliary atresia

20. Crigler-Najjar syndromes
• Due to mutations in the gene encoding bilirubin UDP-glucuronosyl transferase
• Leading to complete absence of this hepatic enzyme activity (Crigler-Najjar syndrome
Type 1) or reduced enzymatic activity (Crigler-Najjar syndrome Type 2)
• Crigler-Najjar syndrome Type 1 : - Severe congenital unconjugated hyperbilirubinemia
( S.Bil > 20mg/dl)
- Kernicterus present
- Often fatal within first 15 months of life
• Crigler-Najjar syndrome Type 2: - Less severe and more benign than Type 1
( S.Bil does not exceed more than 20mg/dl)

21. Gilbert syndrome
• Due to mutations in the gene encoding bilirubin UDP-glucuronosyl transferase
• About 30% of the enzymatic activity retained
• Resulting in congenital unconjugated hyperbilirubinemia; condition is harmless

22. Dubin-Johnson syndrome and Rotor syndrome
• Rare Autosomal recessive disorder
• Rotor syndrome less frequent than Dubin-Johnson syndrome
• Due to mutations in the gene encoding for the bile canalicular membrane protein involved
in secretion of conjugated bilirubin into bile
• Both diseases are clinically similar resulting in mild conjugated hyperbilirubinemia,
however with one major difference that liver in patients with Rotor syndrome has no
increased pigmentation and appears totally normal

23. Neonatal physiological jaundice
• Unconjugated hyperbilirubinemia is present
• Results from accelerated hemolysis and an immature hepatic system for the uptake,
conjugation and secretion of bilirubin
• If plasma concentration of unconjugated bilirubin exceeds >20-25mg/dl and if left
untreated, can result in kernicterus
• Exposure of jaundiced neonates to phototherapy promotes conversion of unconjugated
bilirubin to more polar and water-soluble isomers.
• These photoisomers can thus further be excreted into the bile.
( Note – Because of solubility differences, only unconjugated bilirubin crosses the BBB
causing kernicterus, while conjugated bilirubin appears only in urine)

24. Refrence : Medscape; Phototherapy for Jaundice

25. HEMOLYTIC ANAEMIA
• Group of disorders characterised by increased RBC destruction (hemolysis)
• RBC lifespan gets reduced from normal 100-120 days
• Anemia may not become evident, condition termed as ‘compensated hemolytic disease’
- Shows marked reticulocytosis
- Erythroid hyperplasia of bone marrow increasing RBC production six to eight fold to
maintain normal Hb levels
• However, when anemia becomes evident, it is termed as hemolytic anemia
• The hemolytic anemias may, therefore, be classified into two broad groups:
- Hemolytic anemias due to a corpuscular defect (intracorpuscular or intrinsic abnormality)
- Hemolytic anemias due to an abnormal hemolytic mechanism (extracorpuscular or
extrinsic abnormality)

26. HEMOLYTIC ANAEMIAS DUE TO A
CORPUSCULAR DEFECT
(INTRACORPUSCULAR OR INTRINSIC
ABNORMALITY)
HEMOLYTIC ANAEMIAS DUE TO AN
ABNORMAL HAEMOLYTIC MECHANISM
(EXTRACORPUSCULAR OR EXTRINSIC
ABNORMALITY)
• These are mainly congenital.
• Can be acquired also.
• Congenital: basic defect in any of three main
components of the cell - the membrane
- the haemoglobin molecule
- the enzymes concerned
with cell metabolism
• These disorders are acquired.
• Hemolysis may result from either an immune or a
non-immune mechanism

27. Classification of Hemolytic Anaemia
INTRACOPUSCULAR (INTRINSIC)
Congenital :
• Membrane defects
a) Hereditary spherocytosis
b) Hereditary elliptocytosis
c) Hereditary xerocytosis and hydrocytosis
d) Hereditary stomatocytosis
e)Abetalipoproteinemia (acanthocytosis)

28. • Hemoglobin defects
a)Hemoglobinopathies: - Sickle-cell anaemia
- Other homozygous disorders (Hb-C, Hb-E, Hb-D, etc.)
- Unstable hemoglobin disease
b) Thalassaemia: Beta-thalassaemia major, Hb-H disease
c) Double heterozygous disorders: Sickle-cell beta thalassaemia, Hb-E beta thalassemia etc.
• Enzyme defects
a)Non-spherocytic congenital hemolytic anaemia
- Deficiency of pyruvate kinase or other enzymes of the Embden- Meyerhof pathway
- Deficiency of glucose-6-phosphate dehydrogenase or other enzymes of the pentose phosphate
pathway
b) Drug-induced hemolytic anemia and favism
Acquired:
- Paroxysmal nocturnal hemoglobinuria (PNH)

29. EXTRACOPUSCULAR (EXTRINSIC)
• Immune mechanisms
a)Auto-immune acquired hemolytic anemia
- Warm antibody
- Cold antibody
b) Hemolytic disease of the newborn
c) Incompatible blood transfusion
d) Drug-induced hemolytic anaemia
• Non-immune mechanisms
a)Mechanical hemolytic anemia:
- Cardiac hemolytic anemia
- Microangiopathic hemolytic anemia
- March hemoglobinuria

30. • Miscellaneous
a) Hemolytic anemia due to direct action of chemicals and drugs
b) Hemolytic anemia due to infection : Protozoal infections like Malarial (P. Falciparum),
babesiosis, toxoplasmosis, leishmaniasis; bacterial like cholera, salmonella, clostridial etc.
c) Hemolytic anaemia due to burns
d) Lead poisoning
e) Venoms
f) Hypophosphatemia
g) Thermal injury

31. RED CELL DESTRUCTION
Destruction of red cells is mainly of two types:
Extravascular hemolysis:
-Site of destruction is mainly RE cells of spleen, liver and bone marrow
- Major mechanism of red cell destruction in physiological state
- Hemoglobin is catabolised to bilirubin which is taken up by liver for conjugation and further
metabolism
- This mechanism gets exaggerated in hemolytic anemias resulting in unconjugated hyperbilirubinemia
Intravascular hemolysis:
- Red cells get destroyed while in circulation
- Osmotic lysis and red cell fragmentation can cause intravascular destruction
- Hemoglobin gets released within the plasma
- Main pathway in pathological states like PNH, G6PD deficiency etc.

32. Ref: GK Pal comprehensive textbook of

33. FATE OF HEMOGLOBIN IN INTRAVASCULAR
HEMOLYSIS
REFRENCE : Wintrobes
clinical hematology
13th edition

34. Haemolytic element in other anaemias:
• Include the anaemias associated with disseminated malignancy, leukaemia, malignant
lymphomas, renal failure, liver disease, rheumatoid arthritis, and the megaloblastic
anaemias.
• Shortening of red cell lifespan is minimal.
• Impairment of red cell production is more important factor in the pathogenesis of the
anaemia.
• Usual clinical features of a haemolytic anaemia are seldom present; jaundice is absent,
serum bilirubin within normal range and reticulocyte count is normal or slightly
increased.

35. Approach to diagnose Hemolytic Anemia
• HISTORY MUST BE TAKEN PROPERLY FOR DETERMINATION OF THE AETIOLOGY OF
THE DISEASE
• The following points in the history should be incorporated:
Age at onset of symptoms : whether the pallor developed at birth/younger age (congenital hemolytic
anemia) or in middle/elderly age (acquired haemolytic anemia)
Sex : G6PD deficiency haemolytic anemia affects male>> females
Race : Some hemolytic anemias (mainly congenital hemolytic anemia) are prevalent in certain races
and regions like:
- Sickle cell in Africa and central part of India
- Hb E disease in Thailand, Burma and eastern part of India
- Thalassemia in Mediterranean countries, South-east Asia; in India higher incidence in races
like Sindhis, Punjabis, Gujaratis and Parsis.

36. Occupation : History of working in certain oxidising chemical factories to rule out miscellaneous
cause of acquired hemolytic anemia
Nature of onset of symptoms : whether the pallor developed sudden (acute hemolysis mostly seen in
acquired hemolytic anemias) or insidiously (chronic hemolysis as seen in congenital cases)
Jaundice; colour of urine and faeces : H/o of episodes of jaundice; whether mild/persistent (sickle
cell anemia) or intermittent attacks (hereditary spherocytosis) or if severe jaundice with clay coloured
stools then hepatocellular and obstructive jaundice should be ruled out
Cholelithiasis : May develop in some cases of congenital hemolytic anemias
Aplastic crises : H/o infection with human parvovirus B19 causing pancytopenia, further worsening
the disease

37. Hemoglobinuria; relation to sleep, cold, exercise, or drugs
For Eg : -Red coloured urine especially in morning hours s/o PNH
-Cola coloured urine s/o intravascular hemolytic reactions due to cold or warm auto-
antibodies / alloantibodies/ drug induced/ exertional (March hemoglobinuria)
-Dark coloured urine in falciparum malaria (Blackwater fever)
Family history : H/o anemia, jaundice, splenomegaly, dark urine, cholelithiasis in family and
also to determine the Autosomal dominant/recessive or sex linked congenital hemolytic
anemias
For Eg : Hereditary spherocytosis – Autosomal dominant
Thalassemia – Autosomal Recessive
Sickle cell disorders - Autosomal Recessive
G6PD – Sex linked

38. Symptoms suggestive of disorders causing secondary AIHA such as lymphoma, lupus,
rheumatoid arthritis etc
History s/o Raynaud's phenomenon
Drugs like primaquine, chloroquine, nitrofurantoin etc may induce hemolysis in G6PD
deficiency
Chemical or alcohol ingestion
Recent travel to tropics
Recent transfusion history

39. • PROPER PHYSICAL EXAMINATION MUST BE DONE AND CLINICAL
MANIFESTATIONS MUST BE LOOKED FOR
• Following features should be looked for:
Pallor, cyanosis: Mild to severe depending upon degree of hemolysis
Jaundice: Mild to moderate, may be severe in neonatal period, mainly of unconjugated type.
Not associated with pruritis.
Splenomegaly: Mild to moderate and may progress with advancing age
 Hepatomegaly
Cholelithiasis: Patients often have s/s of gall bladder disease; having black pigment stones

40. General development, facies, presence of leg ulceration, or pigmentation
Skeletal changes: Skeletal abnormalities like tower shaped skull, thickening of flat bones,
distortion of maxillary sinuses, dental abnormalities and growth retardation
Aplastic crisis: Following Human Parvovirus B19 infection, exacerbating the course of
disease, causing severe anemia and pancytopenia
Signs of disorders causing secondary AIHA, especially lymph node enlargement and purpura
Colour of urine : - Cola coloured urine s/o intravascular hemolysis
- Dark coloured urine in falciparum malaria induced hemolysis
-Red coloured urine especially in morning hours s/o PNH

41. Ref : De Gruchy's Clinical Haematology in Medical Practice, 6th edition

42. FOLLOWING ESSENTIAL INVESTIGATIONS MUST BE CARRIED OUT IN
ALL SUSPECTIBLE CASES OF HEMOLYTIC ANEMIA :
• Full blood examination, with special reference to:
• CBC : ↓ Hb levels, ↑ reticulocyte count (5 - 20%) ( Normal range 0.2-2%) due to erythroid
hyperplasia in bone marrow ( hallmark of various haemolytic anemias)
• Morphology of red cells in a well-stained blood film should be observed; note for:
• Polychromatic macrocytes : due to pre-mature delivery of reticulocytes into the
circulation
REFRENCE : Wintrobes
clinical hematology
13th edition

43. • Spherocyte : Spheroid RBC with no central pallor seen in hereditary spherocytosis,
immunohemolytic anemia and burns
REFRENCE : Wintrobes
clinical hematology
13th edition

44. • Elliptocytes : Oval RBC seen in Hereditary elliptocytosis and megaloblastic anemias
REFRENCE : Wintrobes
clinical hematology
13th edition

45. • Stomatocytes : Uniconcave red cells; slit like rather than circular area of central pallor
seen in Hereditary stomatocytosis and alcoholism
REFRENCE : Wintrobes
clinical hematology
13th edition

46. • Acanthocytes : 5-10 spicules of various lengths; irregular in spacing and thickness seen
in spur cell anemia with liver disease

47. • Echinocytes/Burr cells : 10-30 spicules evenly distributed over cell surface as seen in
case Uremia

48. • Sickle cells : RBC with sickle shape as in case of Sickle cell anemia
REFRENCE : Wintrobes
clinical hematology
13th edition

49. • Target cells : Solid area in center of central pallor seen in Thalassemia, Hemoglobin C
disorders, liver disease, postsplenectomy
REFRENCE : Wintrobes
clinical hematology
13th edition

50. • Schistocytes : Triangular, helmet-shaped,fragmented or greatly distorted cell seen in
microangiopathic haemolytic anemias, in cases of turbulent blood flow etc
REFRENCE : Wintrobes
clinical hematology
13th edition

51. • Auto-agglutination : As seen in Cold Agglutinin Disease (CAD)
REFRENCE : Wintrobes
clinical hematology
13th edition

52. BIOCHEMICAL INVESTIGATIONS
• Total serum bilirubin level : Raised (mainly unconjugated) (Normal range: 0.2-1mg/dl)
REFRENCE : Wintrobes
clinical hematology
13th edition

53. • Serum Lactate Dehydrogenase (LDH) level : Raised (more in intravascular hemolysis) (Normal
range: 105-330 IU/L)
• Serum haptoglobin level: Normal in extravascular but decreased in intravascular hemolysis
(Normal range: 40-200 mg/dl)
REFRENCE : Wintrobes
clinical hematology
13th edition

54. • Examination of plasma for hemoglobin, hemopexin and methemalbumin
• Examination of urine for urobilinogen, hemoglobin, hemosiderin
IMMUNO-HEMATOLOGICAL INVESTIGATIONS
• Direct antiglobulin test : confirms in vivo sensitisation of red cells with auto or allo-antibodies
• Antibody screening : to confirm the antibody

55. APC Textbook of Haematology

56. • FURTHER SPECIAL HEMATOLOGICAL AND RADIOLOGICAL INVESTIGATIONS
NEEDED TO BE CARRIED OUT
• These include:
• Bone marrow aspiration and trephine biopsy (evidence of lymphoma, folate deficiency in AIHA)
• Measurement of red cell lifespan test (establish haemolytic nature in doubtful cases)
• Osmotic fragility test : confirms the presence of spherocytes and elliptocytes
• Sickling test (sickle-cell anaemia)
• Tests for abnormal haemoglobins: haemoglobin electrophoresis alkali denaturation, high-pressure
liquid chromatography, Hb-H inclusions, heat instability test

57. • Examination of red cells for methaemoglobin and sulphaemoglobin (chemical haemolytic
anaemia)
• Heinz-body preparation (chemical haemolytic anaemia, hereditary haemolytic anaemia)
• Estimation of red cell G6PD and other enzymes for screening and assays (hereditary
haemolytic anaemia, chemical haemolytic anaemia)
• Cold agglutinins (AIHA)
• Investigations to demonstrate aetiology in secondary AIHA, especially LE cell test and
lymph node biopsy
• Investigation of relatives (for hereditary haemolytic anaemia)

58. • VDRL test (paroxysmal cold haemoglobinuria)
• Donath-Landsteiner test (paroxysmal cold haemoglobinuria)
• Ham's acid serum test, sucrose haemolysis test (paroxysmal nocturnal haemoglobinuria)
• Radiology: - X-ray of skull, hands, and long bones (hereditary haemolytic anaemia)
- Ultrasound of abdomen (to look for splenomegaly and hepatomegaly)
- Computed tomography of chest and abdomen

59. Ref : De Gruchy's Clinical Haematology in Medical Practice, 6th edition

60. REFRENCES
• Harper’s Illustrated Biochemistry 31st edition
• Harrison’s Principles of Internal Medicine, 18th edition
• Lippincott Illustrated Reviews of Biochemistry 7th edition
• De Gruchy’s Clinical Haematology in Medical Practice, 6th edition
• Wintrobe’s Clinical Hematology 13th edition
• APC Textbook of Haematology